Cryptococcus neoformans is a deadly fungal pathogen that exhibits pronounced neurotropism: it is the most common cause of fungal meningitis, particularly in immunocompromised patients, resulting in over 620,000 deaths annually. How C. neoformans cells traverse the blood-brain barrier (BBB) to infect the central nervous system (CNS) remains poorly understood. Our recent studies implicate inositol ? one of the most abundant metabolites in the brain ? in this process. In particular, we find that growth of C. neoformans under inositol-rich conditions enhances fungal virulence and that fungal mutants defective in inositol uptake exhibit reduced virulence, reduced capacity to transmigrate from the blood into the brain, and reduced ability to traverse a model BBB in vitro. We also find that C. neoformans compromises tight-junction integrity in vitro, promoting inositol leakage through the brain microvascular endothelial monolayer. Furthermore, we show that inositol induces the expression of fungal cell surface factors involved in virulence, including the polysaccharide capsule, a major fungal virulence factor, and hyaluronic acid (HA), a ligand important for fungal binding to the BBB. Finally, we find that growth on inositol promotes the production of capsule structures involved in immune evasion and that, conversely, C. neoformans mutants defective in inositol uptake elicit enhanced protective immunity during brain infection. Based on these results, we hypothesize that C. neoformans senses and utilizes host inositol to modify the fungal cell surface in a way that promotes penetration of the BBB and development of cryptococcal meningitis. Interestingly, C. neoformans contains an unusually complex inositol uptake system and catabolic pathway, which likely evolved from its utilization of the inositol stores of its plant reservoirs. Thus, this fungus may be uniquely adapted to thrive in the inositol-rich environment of the CNS and to utilize inositol-dependent pathways for pathogenesis. The overarching goal of this proposal is to obtain a detailed understanding of the mechanism by which C. neoformans acquires and utilizes host inositol to establish human brain infection. We propose three Specific Aims: 1) Define inositol sensing and metabolic pathways required for modifying fungal cell surface structure by using a combination of fungal mutagenesis analysis, enzymatic assays, and polysaccharide structural analysis; 2) Characterize the mechanism of inositol- mediated promotion of C. neoformans BBB crossing and CNS infection by using an in vitro BBB model system and in vivo animal models; and 3) Define the transcriptional circuits regulating inositol-dependent processes during cryptococcal brain infection by analyzing the expression and localization of fungal inositol factors and identifying transcription factors regulating their expression during infection. Together, these studies will elucidate a novel contribution of a brain metabolite, inositol, to the development of life-threatening fungal meningitis. As such, these studies promise to provide substantial insight into mechanisms by which pathogens cross the BBB and establish CNS infections.